The chemistry, physiology and pharmacology of the alpha-melanocyte stimulating hormone (alpha-MSH) system was studied. By combining high pressure liquid chromatography with a radioimmunoassay, alpha-MSH and deacetylated alpha-MSH and their respective methionine sulfoxide forms, which are formed artifactually, were identified. By studying the effects of these peptides on behavior, it was determined that deacetylated alpha-MSH was an inactive form and probably a precursor to alpha-MSH. Alpha-MSH is released in vitro by veratridine or potassium stimulation in a calcium dependent manner. Stimulation of the central gray causes in vivo release of alpha-MSH into the cerebrospinal fluid. An acute but not chronic stress also causes a dramatic decrease in brain alpha-MSH concentrations which is interpreted to indicate a release of alpha-MSH. A diurnal rhythm of alpha-MSH in the brain was shifted by clorgyline treatment. Alpha-MSH concentrations in the brain were decreased by fluphenazine treatment and increased by housing rats in chronic dark.